Decreased serum vitamin D (VD) levels have been associated with gastrointestinal (GI) disorders, including irritable bowel syndrome (IBS). VD can also modulate the intestinal barrier. Given the link between the GI barrier’s alterations and diet, attention has aroused the positive effects of the Low FODMAP Diet (LFD) on IBS patients’ symptom profile. We evaluated the GI symptoms and the urinary and circulating markers of GI barrier function, the markers of inflammation and intestinal dysbiosis in 36 IBS patients with predominant diarrhea (IBS-D)  categorized for their circulating VD levels in low (L-VD) and normal (N-VD) (cutoff = 20 ng/mL). Evaluations were performed before and after 12 weeks of LFD. At the baseline, L-VD patients showed a significantly worse symptom profile and altered small intestinal permeability (s-IP) than N-VD. After LFD, a significant increase in the circulating VD levels in both the subgroups and a significant improvement of s-IP in L-VD patients occurred. Finally, VD levels negatively correlated with the symptom score and fecal zonulin. These data highlight the close relationship between VD and the intestinal barrier and support their involvement in IBS-D pathophysiology. Moreover, the potentially positive role of LFD in the management of IBS-D was confirmed. Vitamin D (VD) belongs to the steroid hormone family, and evidence is accumulating on its influence on human health homeostasis . VD is eThe major circulating form of VD is 25-hydroxy VD [25(OH)D], which is also used to assess VD status in clinical practice . There iA considerable percentage of the IBS population can be VD deficient, and, recently, the successful treatment of diarrhea-predominant IBS (IBS-D) and its associated symptoms, with high oral doses VD supplementation, has aroused the attention for this hormone as a therapeutic option for the IBS-D management . IntolerVD also has shown potentialities in modulating the intestinal barrier , and theOther tests to evaluate the GI barrier function include the fecal and serum levels of zonulin, a human protein that reversibly regulates s-IP by changing the TJ interaction . AdditioMany IBS patients often describe a worsened symptom profile in consuming certain foods, such as those containing the so-called fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) . ConsequBased on these premises, our research was designed to investigate the relationship between VD, evaluated as 25(OH)D, s-IP, and the integrity of the GI barrier in IBS-D patients following a long-term (12 weeks) LFD. The main aim of this study was to compare the GI symptoms, assessed by the IBS symptom severity scale (IBS-SSS)  and the IBS-D patients, diagnosed according to the Rome IV criteria, were recruited from January 2018 to September 2019, among the outpatients of the Nutritional Physiopathology Laboratory—National Institute of Gastroenterology “S. de Bellis” Research Hospital located in Apulia, a Mediterranean region in south-eastern Italy.After a physical examination, patients completed the “Gastrointestinal Symptom Rating Scale” (GSRS) questionnaire , provideThe female patients provided the blood and urinary samples during the follicular phase  to avoid interference and contamination of the urine samples with blood. The inclusion criteria were age 18 years or older; symptoms resembling IBS-D for at least 14 days; a stool pattern, as described by Schmulson et al. ; a minimThe exclusion criteria were: pregnancy; intense physical activity; constipation; fever; a diagnosis of post-infectious IBS, giardiasis, previous abdominal surgery, endocrine and metabolic disorders; cardiovascular diseases; altered hepatic and renal functions; secondary causes of intestinal atrophy, previous diagnosis of neoplasms; the use of drugs for alleviating IBS symptoms or probiotics in the two weeks before evaluation; previous antibiotic therapy; the use of selective serotonin reuptake inhibitors or other antidepressant drugs. The reasons for study interruption  were recorded in the case report form. Written informed consent was obtained from all the subjects to collect anthropometric values, clinical data, and analytical measurements.To avoid possible bias due to the exposure to sunlight, the enrollment of patients was suspended during the summer months.http://www.clinicaltrials.gov (NCT03423069)-last accessed data: 17 February 2021.This study was part of a research project approved by the local Scientific Committee and the Institutional Ethics Committee of IRCCS Ospedale Oncologico—Istituto Tumori Giovanni Paolo II, Bari, Italy, (N. 274/C.E. 12.12.17), and it was part of a registered clinical trial on The study design included three visits.Visit 1 (Baseline). The subjects underwent a gastroenterological examination and received verbal and written information about the study. They gave their informed consent about the goals of this study to evaluate the efficacy of a diet intended to reduce IBS symptoms and be followed for 12 weeks. Then, subjects underwent an interview with trained nutritionists to obtain information on their lifestyle, physical activity, dietary habits, physiological and pathological conditions. The eligible patients were invited to consume their usual diet and fill in a daily diary of their food habits for the following seven days. The diary included the record of the stool characteristics based on the intestinal habits, the Bristol stool form chart , the useVisit 2 (Diet attribution). One week after visit 1, the anthropometric measurements were performed. All the patients completed the IBS-SSS questionnaire . They haVisit 3 . After 12 weeks of the diet, the researchers collected the symptoms and food questionnaires filled in the previous days. The patients received the IBS-SSS and a food diary for checking adherence to the diet . All the patients underwent the same procedures again as at visit 2 for the anthropometric measurements and analytical measurements.The IBS-SSS, a validated GI symptoms questionnaire , was useThe patients had to record a food diary at the start of the study and during the diet intervention to evaluate their energy intake and energy consumption. The diary included details of the quantities (expressed in grams) and the types of food consumed daily at breakfast, lunch, dinner, during snacks, and the type of physical activity and duration .http://www.progettodieta.it-last accessed data: 18 March 2020). The daily energy intake and consumption expressed in kcal, the percentage and weight of daily carbohydrates, lipids, proteins, the percentage of alcohol, the weight of the dietary fiber, minerals, and vitamins, were calculated.Nutritionists evaluated food diaries completed before and during the diet period. All data were analyzed by dedicated software  chlorohexidine was added to each collection as a preservative regardless of the final volumes. The total urine volumes from individuals were measured and recorded. After thoroughly mixing, a portion of 2 mL was taken and stored at −80 °C until examined. The detection and measurement of the three sugar probes, La, Ma, and Su in urine, were performed by chromatographic analysis, as described previously by our group [All the participants in the study underwent s-IP evaluation by SAT after fasting overnight. Pretest urine was collected in our laboratory to check for the possible presence of endogenous sugars. Then subjects drank a sugar test solution containing 10 g of La, 5 g of Ma, and 40 g of Su in a volume of 100 mL. Urine samples were collected up to 5 h after administration. A 1 mL volume of 20%  anticoagulant. Raw stool samples from the IBS-D patients were frozen and stored at −80 °C within 12 h after the sampling. Before the laboratory analysis, stool samples were thawed, and mechanical homogenization was performed using an inoculation loop. The Fecal Sample Preparation kit  to prepare fecal eluates was used. VD status was quantitatively assessed by measuring serum 25(OH)D levels using a chemiluminescence system . Serum and fecal zonulin were assayed by ELISA kits . Following the manufacturer’s instructions, serum and fecal levels were considered normal for values lower than 48 ng/mL and 107 ng/mL, respectively. I-FABP and DAO serum concentrations were evaluated by ELISA kits  and , respectively. IL-6 and IL-8 circulating levels were measured by ELISA kits . Lipopolysaccharide (LPS) was assayed using an ELISA kit by Cloud-Clone Corp. .All patients collected a sample of urine in the morning. A standard colorimetric assay kit  was used according to the manufacturer’s urinary indican determination procedures. The detection and measurement of skatole in urine were performed by the 3-methylindole kit  on a Thermo Scientific model Dionex high-performance liquid chromatography (HPLC) system consisting of an UltiMate 3000 pump and a Rheodyne injector with a 20-μL loop . Samples, calibrators, and quality controls were prepared according to the manufacturer’s instructions. In detail, 950 μL of buffer reagent and 20μL of the internal standard were added to 50 μL of a urinary sample. After vortexing, 20 μL of urine samples were injected into the HPLC system. A Poroshell 120 EC-C18 column  and a mobile phase flow rate of 1.0 mL/min was used for skatole separation. The sample run was 15 min, and spectrofluorimetric detector wavelengths were set at 280 nm (excitation) and 360 nm (emission). Urinary indican and skatole values higher than 20 mg/L and 20 µg/L are considered indices of fermentative and putrefactive dysbiosis, respectively .t-Values and their significance level were calculated to test the hypothesis that the contribution (the regression coefficient) of an entered variable significantly differed from zero. Pearson’s correlation was performed comparing VD versus all other variables. All the differences were considered significant at a 5% level. A specific statistical package  was used.All results are expressed as means ± SEM unless otherwise specified. Nonparametric tests were performed to avoid violation of the assumption of normal distribution. The Wilcoxon rank-sum test was used to detect differences between the items of the IBS-SSS questionnaire and the biochemical parameters before and after the LFD in the IBS-D patients considered as a whole group or subgroups categorized according to low (L-VD) or normal (N-VD) circulating VD concentrations at the start of the study. The Mann–Whitney test was applied in comparing the two subgroups before and at the end of the diet. Linear regression analysis was performed, considering VD as the dependent variable, and those clinical and biochemical parameters significantly correlated with VD as independent variables in a stepwise regression procedure. The explained variance (adjusted R-squared) was determined for the regression, and it was tested with the F-test. The patients’ anthropometric characteristics and serum biochemical parameters at baseline and the end of the diet are summarized in Both the protein and carbohydrate percentages significantly increased after LFD in comparison to baseline. By opposite, lipid grams and percentage, as well as the total FODMAP content, significantly decreased at the end of the diet compared to the start of the study. No significant difference in mineral and vitamin levels was observed.p < 0.0001) by approximately 28% (25 ± 2.4 vs. 32 ± 2.2 ng/mL) at the end of dietary treatment compared to baseline.In the whole group of IBS-D patients, the VD levels increased significantly  had VD levels lower than 20 ng/mL (L-VD); twenty out of 36 patients (55.5%) had normal VD levels, higher than or equal to 20 ng/mL (N-VD).p < 0.0001) compared to baseline (15.2 ± 0.98 ng/mL), reaching values indicative of sufficient circulating amounts of the hormone. The N-VD patients also experienced a significant (p = 0.028) increase in VD concentrations after LFD (36.2 ± 3.4 ng/mL vs. 32.5 ± 3.4 ng/mL), although at a less extent (+11.4%). Despite the significant rise in VD levels in the L-VD subgroup at the end of LFD, the concentrations of 25(OH)D remained significantly lower (p = 0.048) than those in the N-VD patients.In the L-VD subgroup, the increase in circulating VD concentrations after the diet (27 ± 2.2 ng/mL) was massive (+80%) and significant  higher in the L-VD patients compared to N-VD ones. However, the LFD improved the symptom profile in the totality of IBS-D patients since all the single items and the total score was significantly reduced by 12 weeks of LFD in the two subgroups. More in detail, “abdominal pain” reduced by 49.15% in L-VD and 57.25% in N-VD; “interference on life” reduced by 38.00% in L-VD and 37.92% in N-VD; “stool frequency”, reduced by 34.78% in L-VD and 28.23% in N-VD. Finally, the “total score” was reduced by 45.97% in L-VD and 50.28% in N-VD.The symptom profiles were then evaluated according to the circulating VD levels at the start of the study. L-VD patients had a significantly worse clinical profile than N-VD patients at the diet’s start. Specifically, the single items relative to intensity and frequency of the “abdominal pain”, “interference on life in general”, “stool frequency”, and the “total score” were all markedly and significantly  lower than the baseline percentages. IBS-D patients after LFD also showed significantly (p = 0.001) lower% Ma values than those at the start of the study. Consequently, the La/Ma ratio reduced significantly (p = 0.012) by 27.6%. Compared to values at the start of the study, a statistically significant (p = 0.033) and sharp (−30.8%) decrease of the excreted sucrose percentage at the end of the diet occurred.At the end of the study, in the total IBS-D patients,% La was significantly (p = 0.002) and the La/Ma ratio (p = 0.002) between the L-VD and N-VD subgroups. Noteworthy, at the start of the study, the L-VD patients, but not the N-VD ones, had a mean La/Ma ratio of 0.042, a value higher than the cutoff (0.030).Categorizing the patients according to low or normal basal VD levels at the start of the diet, the Mann–Whitney test revealed a significant difference in% La (p < 0.0001) reduced by 54% the recovery percentages of lactulose in the L-VD subgroup. N-VD patients showed a significant (p = 0.025) reduction by 11.7% in the percentage of mannitol recovery compared to baseline values. In response to LFD, the La/Ma ratio significantly (p = 0.039) decreased by 31% in L-VD patients, reaching a value far below the cutoff (0.023) and similar to that in N-VD patients (0.020). Finally, the urinary excretion of sucrose was reduced by LFD in both L-VD (−43%) and N-VD (−24%) groups, although without reaching a statistical significance.After LFD, the nutritional treatment significantly  are reported in p = 0.048) lower fecal zonulin concentrations (134.4 ± 10.12 ng/mL) than those at the start of the diet (162.22 ± 12.66 ng/mL). In addition, serum zonulin values significantly decreased at the end of the diet . As for the intestinal barrier integrity, the post-diet concentrations of I-FABP and DAO were significantly (p < 0.001) lower than the pre-diet concentrations in the whole IBS-D group.The fecal zonulin concentrations of the whole group of IBS-D patients were far above the cutoff level of 107 ng/mL, irrespective of VD levels and the nutritional treatment. As regards the effect of diet, the total group of IBS-D patients had significantly . LFD significantly reduced the levels of fecal zonulin (p = 0.019), I-FABP (p = 0.016). and DAO (p = 0.036) in L-VD patients. Moreover, LFD significantly (p = 0.008) reduced only the I-FABP concentrations in the N-VD subgroup.There was no difference in the values of all the above markers at the start of the diet between L-VD and N-VD subgroups, except for fecal zonulin . No difference in the inflammatory profile was found between NVD and LVD subgroups either before or after the diet.The circulating levels of IL-6 and IL-8 in the IBS-D patients are reported in p = 0.032) of the indican urinary concentration was observed at the end of LFD in the total IBS-D patients. When the patients were categorized according to VD levels at baseline, no difference was found between N-VD and L-VD. Comparing the indican concentration before and after LFD, no difference was found in both the L-VD subgroup and the N-VD one. Similarly, no difference was found comparing N-VD and L-VD patients at the end of the diet  , although a significant decrease was observed after diet in the total group (= 0.022) , panel Bp = 0.010) at the start of the study (0.050 ± 0.01 ng/mL) compared to the values at the end of the diet (0.043 ± 0.02 ng/mL). After categorizing for VD levels, L-VD subgroup (0.049 ± 0.002 ng/mL vs. 0.042 ± 0.003 ng/mL), but not the N-VD one (0.044 ± 0.002 ng/mL vs. 0.042 ± 0.02 ng/mL) showed significantly (p = 0.042) reduced levels of LPS at the end of the diet compared to baseline.Finally, in the total group of IBS-D patients, the LPS concentrations were significantly higher (p = 0.004), VD levels and fecal zonulin . A trend toward significance between VD and La , and VD and LPS was finally found . (r = Pearson’s correlation coefficients).Considering all the examinations per each variable, a negative correlation was found between VD levels and IBS-SSS total score    . These dData obtained in this study highlight the close relationship between VD and the intestinal barrier and support the involvement of VD deficiency in the IBS-D pathophysiology. Moreover, the potentially positive role of LFD in the management of IBS-D is further confirmed.There is massive evidence that the VD deficiency not only has a negative impact on the human skeletal system and the progression of multiple diseases  but also of gut diseases, including colorectal cancer and IBD . The latIn our study, at the start of the diet, the whole group of IBS-D patients showed a mean VD concentration only slightly higher than the cutoff value of 20 ng/mL. This vitamin content is now widely accepted as adequate for bone and overall health in healthy individuals . When paFrom a clinical perspective, L-VD patients also had a significantly higher symptom profile at IBS-SSS than N-VD ones. The VD concentrations appeared to depend on both the symptom profile and the state of health of the intestinal barrier, as linear regression analysis showed. The present findings are in line with the reported association of low circulating VD levels with increased central sensitivity, chronic pain , depressAs regards the effect of the LFD on IBS, different data in the literature indicate that restricting FODMAP consumption could be beneficial for symptoms and intestinal barrier function ,44, and Significant decreases in weight, BMI, abdominal and waist circumferences were observed after LFD. Weight loss and the reduction of BMI were not among the goals of our present study. However, these reductions were probably due to the duration and restrictions of the diet. Weight loss and BMI reduction can represent a possible consequence of a long-term, personalized diet, even if the energy intake during LFD was not significantly reduced. It is known that the “elimination phase” leads to the reduction of body weight and BMI, following the caloric restriction of some foods high in FODMAPs .Previous reports proved that LFD did not modify VD levels in either IBS patients or CD patients treated in combination with a gluten-free diet ,47. In tInterestingly, when the IBS-D patients were categorized according to the VD status, L-VD patients responded better to diet than N-VD patients. In particular, VD levels increased by 41% in the former group and reached normal values at the end of treatment. While on one side, these results indicate a possible therapeutic role in the control of IBS for VD, on the other, let us hypothesize that dietary components able to alleviate IBS symptoms can also affect VD levels via changes in intestinal permeability. In this context, the s-IP expressed as the urinary La excretion and the La/Ma ratio decreased by 54% and 45%, respectively, in L-VD and IBD-D( + ) at the end of the study, reaching values indicative of a normal s-IP. LFD in L-VD patients concordantly reduced fecal zonulin levels and circulating I-FABP and DAO levels, highlighting improved epithelium integrity. This amelioration resulted in a lower translocation of Gram-negative bacteria throughout the intestinal villi, as evidenced by the lower circulating levels of LPS after diet . On the The present research has some weaknesses. First, VD specific receptor was not evaluated. Thus, we could not obtain complete information about the actual processes occurring at a mucosal level for the vitamin/VDR complex. Second, the cohort of patients was too small to draw firm conclusions. Therefore, further research is needed to investigate the still unveiled relationship between FODMAP, VD levels, and intestinal barrier in IBS. Although our current results and available evidence in the literature support this connection, it is unclear what is causing what. Is VD deficiency causing IBS or, rather, is IBS responsible for VD deficiency? Probably, also other still unknown factors should be taken into account as determinants for both problems. However, based on present and other available data, it seems conceivable that LFD can improve the IBS symptoms by affecting those factors involved in this functional GI disorder, including VD levels and their supposed effects on the integrity and the function of the intestinal barrier.


The PSf/PSf-co-SBPSf blend membranes with various zwitterionic SBPSf segment contents exhibited better surface hydrophilicity and excellent antifouling ability compared to PSf and PSf/PEG membranes. The significant increase of both porosity and water permeance indicates that the PSf-co-SBPSf has a pore-forming effect. The pure water flux and flux recovery ratio of the PSf/PSf-co-SBPSf blend membranes were both remarked to improve 286.43 L/m2h and 92.26%, while bovine serum albumin (BSA) rejection remained at a high level (97.66%). More importantly, the water flux and BSA rejection see minimal variance after heat treatment, indicating excellent thermostability. Overall, the PSf/PSf-co-SBPSf blend membranes achieved a comprehensive performance of sustainable hydrophilic, high permeation flux, and remarkable antifouling ability, thus becoming a promising candidate in high-temperature separation application.Membrane fouling has been one of the most important challenges in membrane separation operations. In this study, we report a facile strategy to prepare antifouling polysulfone (PSf) UF membranes by blending amphiphilic zwitterion polysulfone-co-sulfobetaine polysulfone (PSf-co-SBPSf) copolymer. The copolymer chemical structure was characterized by  Membrane filtration is a green separation method with numerous applications due to its energy efficient, environmentally friendly and modular separation in comparison with other separation process ,2,3. AmoTo achieve the above pursuit, various strategies, such as surface modification, feed pretreatment and innovation, have been adopted to alleviate membrane fouling ,20,21,22Zwitterions have emerged as a new generation of antifouling materials because of their strong intrinsic hydrophilicity, which forms a hydration layer through electrostatic interaction between water molecules and zwitterions ,32,33. T1HNMR), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and water contact angle. The effects of zwitterionic SBPSf segments on membrane morphology, separation performance and antifouling properties were also systematically investigated. To our best knowledge, the amphiphilic copolymer, with polysulfone-based bulk zwitterion and blended with polysulfone, will be rare and very promising. The amphiphilic polymer-blended PSf membranes are expected to have low fouling propensities and thermostability.In this work, a new zwitterionic amphiphilic copolymer polysulfone-co-sulfobetaine polysulfone (PSf-co-SBPSf) was synthesized via a facile synthesis route, and a series of charge-modified polysulfone-based antifouling membranes with blending PSf-co-SBPSf were prepared by the non-solvent-induced phase separation (NIPS) method . The zwi2CO3, ≥99%) were obtained from Aladdin. The 4,4-difluorodiphenylsulfone (DFDPS), 1,3-propane sultone, bovine serum albumin , deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl3) were purchased from Sigma-Aldrich. N-Methyl-2-pyrrolidone (NMP), toluene, formaldehyde, Na2HPO4·12H2O, KH2PO4·2H2O were all purchased from Aladdin. Polysulfone  was provide by Solvay, China.Bisphenol A , dimethylamine (40%), polyethylene glycol , potassium carbonate  were added into the three-necked flask. Subsequently, NMP (80 mL) and toluene (50 mL) were introduced as a solvent and azeotrope reagent. The reaction was heated under reflux at 120 °C for 4 h while the toluene-water was removed from the reaction mixture. Next, the reaction was gradually heated to 150 °C and further reacted for another 8 h. The reaction mixture was cooled to room temperature and poured into deionized water. The polymer was washed with deionized water and ethanol several times. Finally, the polymer was dried at 100 °C under vacuum for 24 h.The PSf-co-TAPSf copolymer was synthesized via traditional step-growth polymerization . The rea3 (100 mL), 1,3-propane sultone  was added. The solution was stirred at 60 °C for 48 h. The copolymers were obtained by rotating evaporation. In addition, to demonstrate the solubility of the zwitterionic copolymer, a different proportion of TAPSF copolymer was synthesized and functionalized by controlling the DABA:BPA ratio in the same fashion as described in To a solution of PSf-co-TAPSf  in CHCl1H NMR spectroscopy. The spectra were recorded on a Bruker 400 MHz NMR  spectrometer using DMSO-d6 and CDCl3 as the solvent.The structures of the pendant tertiary amine functionalized bisphenol A, PSf-co-TAPSf and PSf-co-SBPSf copolymers were characterized by PSf/PSf-co-SBPSf UF membranes were prepared by non-solvent-induced phase separation (NIPS). In a typical process, a certain amount of PSf-co-SBPSf  was well-dispersed into NMP with ultrasonic treatment for 30 min, respectively . Then, the uniform suspension was mixed with PSf (15%) under continuous stirring at 60 °C for 24 h. Meanwhile, pristine PSf (M0) and blended membrane with PEG (M1) were prepared and compared regarding properties. All casting solution was kept in a vacuum oven to remove air bubbles at 60 °C for 24 h. After degasification, the homogeneous dope solution was cast on a glass plate with 150 µm thickness, and subsequently immersed into a coagulation bath for the wet-phase inversion step. Finally, the resulting UF membranes were kept in deionized water for at least 24 h to remove the residual solvent. The various compositions of all membrane dope solutions are summarized in The cross-section morphology of the prepared blending membrane was observed by scanning electron microscopy . Membrane samples were freeze-fractured using liquid nitrogen for cross-sectional examination, and sputter coated with gold before imaging.E = 1486.6 eV) as a radiation source (the take-off angle of photoelectron was set to 90°). The N 1s core-level peaks of hybrid membranes were fitted by using a Lorentzian–Gaussian mixed function.The surface chemical composition of the blended membrane was analyzed by X-ray photoelectron spectroscopy  using Al Kα . The test was performed by depositing 2 μL water drops on the top surface of the dry membrane. Five random spots were measured for each membrane at room temperature and the average value was taken.ε is the porosity, m1 is the weight of the wet membrane (g), m2 is the weight of the dry membrane (g), wρ is the water density , and pρ is the polymer density .The porosity and pore size of each membrane was determined through the dry–wet method ,42. Membmr) was calculated by the Guerout–Elford–Ferry Equation [ε is the porosity (%), μ is the water viscosity (8.9 × 10−4 Pa s), l is the membrane thickness (m), J is the flux (m3 h−1 m−2), and ΔP is the operation pressure (Pa).Furthermore, the mean pore size rm2. The membrane samples were pre-compacted with deionized water for 30 min at a pressure of 0.1 MPa to reach a stable flux. Upon stabilization, pure water flux was collected at 5 min intervals for an hour at a pressure of 0.1 MPa and the average water flux was recorded as w,1J, which was calculated using Equation (3):w,1J is the pure water flux (L m−2 h−1), V is the volume of water permeated (L), A is the effective membrane area (m2) and ∆t is the permeation time (h).Filtration experiments were performed on 25 mm diameter membranes using a 150 mL dead-end filtration cell with an effective filtration area of 12.56 cmJp. The BSA rejection ratio (R) was determined using the following Equation (4):C0 and C1 are the BSA concentrations in feed and permeate solutions (mg/mL), respectively, as measured by a UV-vis spectrometer  at a maximum absorption at 280 nm.To characterize the rejection performance of UF membranes, we used bovine serum albumin (BSA) as the foulant solution during filtration tests. A 1.0 g/L BSA solution  was filtered through the membrane immediately after the pure permeance test. The BSA rejection was collected at 5 min intervals for an hour at 0.1 MPa and the BSA flux was recorded as w,2J to determine the flux recovery ratio (FRR). Each membrane was measured during 3 cycles to evaluate the antifouling performance. The FRR was calculated using the following Equation (5):After BSA solution filtration, the membrane was rinsed with deionized water for 30 min. Then, pure water flux was measured again and recorded as tR), reversible fouling ration (rR), irreversible fouling ration (irR) were calculated using Equations (6)–(8):To further analyze the membrane fouling resistance process in detail, the total fouling ratio  and (2). As shown in The surface chemical composition of UF membranes plays an important role in enhancing hydrophilicity and antifouling. In order to further confirm the surface enrichment of zwitterionic hydrophilic segment, the surface chemical compositions of the pristine PSf membrane and the blend membranes were characterized by XPS. As shown in Surface hydrophilicity was one of the most important factors affecting the membrane antifouling. Water contact angle (WCA) measurement was used to characterize the surface wettability and hydrophilicity. Typically, a smaller contact angle value corresponds to a better hydrophilic surface . Figure 2h, and the BSA rejection was 95.43%. However, the pure water flux and BSA rejection of blend membranes were greatly enhanced with the increase of PSf-co-SBPSf copolymer concentration. For the M5 membrane, the water fluxes up to 286.43 L/m2h, which was almost five times that of the pristine PSf membrane, and the BSA rejection exhibited a slight increase (97.66%). This could be ascribed to the increase in the membrane porosity and the decrease in the thickness of the dense skin layer. Furthermore, the trade-off relationship between permeability and selectivity was broken by blending PSf-co-SBPSf copolymers. This is due to the fact that the zwitterion amphiphilic copolymer tends to produce more narrow pore channels, as summarized in To compare separation performance of the pristine PSf membrane and blend membranes, filtration experiments of pure water and BSA solution were performed. As shown in 2h, while the average BSA flux is only 103.26 L/m2h. Then, the membranes were cleaned for 30 min with water and filtrated with pure water once again. The pure water flux of all membranes after cleaning could not be restored completely to its initial value after three cycles of filtration tests. This is due to the fouling caused by the deposition and adsorption of the BSA onto the membrane surfaces and within the membrane pores, which were difficult to clean completely by washing. However, the pure water flux recovery for the blend membranes was found to be greater than the pristine PSf membrane, showing the higher antifouling performance.Anti-fouling performance is an important property of UF membranes in the separation and purification, which indicates the reusability. The fouling measurement was performed by the three-cycle filtration experiments. The resulting time-dependent flux is shown in t, Rr and Rir values were calculated, respectively, and presented in t values between PSf/PSf-co-SBPSf and PSf/PEG blend membranes, and the values reduced more as the zwitterion concentration increase. That could be attributed to greater coverage of sulfobetaine groups on the membrane surface. Additionally, the Rr value of all blend membranes is higher than pristine PSf membrane, and as high as 57.6% for M5 membrane, which weakens interactions between foulant and membrane surface and could be removed by simple washing as a result of antifouling and reusability [ir of the blend membrane M5 is 7.74%, which is also dramatically lower than that of the pristine PSf membrane, at 57.96%. A relatively lower Rir, the reusability of blend membranes, was further proven [ir values. The structure of the copolymer provides long-term stability of the membranes and also confers excellent hydrophilicity and fouling resistance to the membranes. The excellent antifouling ability of the blend membranes was further illustrated with antibacterial tests. SEM images show that more E. coli bacteria adhered on the surface of the pristine membrane. However, only a few bacteria can be observed on the surface of the blend membranes  for a certain time. The values of water contact angle, water flux and BSA rejection of heated membranes were record with time. It has been reported that when polymer matrix was blended with PEG, there is inevitable leakage from the membrane matrix during separations, resulting in reduced hydrophilicity . As showIn this work, we reported a facile approach to prepare novel polysulfone-based ultrafiltration membranes via NIPS with antifouling and thermostability properties by blending PSf-co-SBPSf copolymer. The zwitterionic PSf-co-SBPSf copolymer was synthesized via the typical Mannich reaction and direct step polymerization method. Compared to the PSf and PSf/PEG membranes, the hydrophilicity, separation performance and antifouling ability of PSf/PSf-co-SBPSf blend membranes were improved remarkably due to the decrease of the skin layer and the increase of the porosity. The result illustrated that the PSf-co-SBPSf could be as a novel porogen, and hydrophilic segment emigrated to the membrane surface during phase inversion. More importantly, the performance of PSf/PSf-co-SBPSf blend membranes has hardly changed after heat treatment. As a control, the PSf/PEG blend membrane exhibited significantly reduced BSA rejection and antifouling ability due to PEG loss at high temperatures. This indicates that the PSf/PSf-co-SBPSf blend membranes have more potential in practical application. Furthermore, the adjusting of the charge content and chain length in the copolymer will provide a great prospect for the antifouling UF membranes. The study provides important research value for the high-temperature application of separation.


Mycobacterium tuberculosis antigens, showing its potential for point-of-care analysis of protein biomarkers.While the bottom-up protein analysis serves as a mainstream method for biological studies, its efficiency is limited by the time-consuming process for enzymatic digestion or hydrolysis as well as the post-digestion treatment prior to mass spectrometry analysis. In this work, we developed an enzyme-free microreaction system for fast and selective hydrolysis of proteins, and a direct analysis of the protein digests was achieved by nanoESI (electrospray ionization) mass spectrometry. Using the microreactor, proteins in aqueous solution could be selectively hydrolyzed at the aspartyl sites within 2 min at high temperatures (∼150 °C). Being free of salts, the protein digest solution could be directly analyzed using a mass spectrometer with nanoESI without further purification or post-digestion treatment. This method has been validated for the analysis of a variety of proteins with molecular weights ranging from 8.5 to 67 kDa. With introduction of a reducing agent into the protein solutions, fast cleavage of disulfide bonds was also achieved along with high-temperature hydrolysis, allowing for fast analysis of large proteins such as bovine serum albumin. The high-temperature microreaction system was also used with a miniature mass spectrometer for the determination of highly specific peptides from  A high-temperature microreaction system is developed for fast and selective hydrolysis of proteins, enabling direct analysis of protein biomarkers by mass spectrometry. Bottom-up proteomics2 is one the most widely used strategies for protein analysis, which usually requires digestion of proteins into peptides by proteases with high sequencing selectivity.3–5 For example, trypsin is widely used for breaking at the C-terminal side of lysine or arginine of peptides under simulative physiological conditions. Most protease-based digestion methods, however, require long-time (∼hours) incubation.6 To accelerate the incubation process, approaches such as microwave-assisted trypsin digestion7,8 and immobilization of trypsin into different materials9,10 have been explored, enabling sufficient trypsin digestion in much shorter time periods.Mass spectrometry (MS) has become a powerful tool for identification of protein primary sequences, post-translational modifications (PTMs) and protein–protein interactions; it also enables determination of various cellular functions at the protein level.11,12 is widely used for MS analysis of digested peptides, which is less sensitive to the matrix effect due to the salts in the samples. However, LC-MS analysis using ESI, which requires sample pretreatment, still plays a major role in proteomic studies, due to better sensitivity, quantitative precision, and sequence coverage obtained with MS/MS analysis of ions at higher charge states.13,14 Although some efforts have been made to accelerate the process of enzymatic digestion, ESI-MS analysis would still be slowed down by the time-consuming purification and post-digestion sample treatment procedures.The enzymatic digestion processes, along with the following enrichment processes, typically require high concentrations of salts in the protein solution, which are not compatible with liquid chromatography (LC)-MS analysis. Extensive sample purification is required to avoid the loss in efficiency for both separation and electrospray ionization (ESI). For this reason, matrix-assisted laser desorption ionization (MALDI)15–19 In this work, we developed a method for selective protein hydrolysis with high efficiency without involving enzymatic digestion with high-concentration salt solutions. A simple procedure was developed using only a weak acid and fast incubation at high temperatures. Protein hydrolysis under strong acidic conditions has been previously used for analysis of proteins and peptides.20,21 It does not involve high-concentration salts in solution but is usually used to recover total amino acids since the sequence information would be lost due to the nonspecific cleavage of peptide bonds.22 Using weak organic acids, selective cleavage at aspartyl residues  in proteins or peptides was achieved.23,24 This process was shown to benefit from high temperatures for incubation but still took two hours to obtain optimal results. Cooper et al.25,26 developed a similar strategy  but without using acids. The hydrolysis process was accelerated by placing the protein solution into a stainless-steel tubing and applying higher temperatures (160 to 207 °C) with an oven. Higher recovery was obtained for model proteins; however, this hydrolysis method was less selective for the breakdown of peptide bonds. These reports testified the effectiveness of the water solution-based high-temperature hydrolysis method for the sequencing of proteins. However, the overall time of the protein analysis is still relatively long (>20 min) due to the requirement of sample pretreatment, pre-conditioning, post-treatment, and multiple-step sample transfer. Recently, in a study of ESI, Chen et al. applied ultrahigh temperatures and high pressures for desolvation and reported the fragmentation of protein and peptide ions.27,28 Inspired by this observation, we developed an in-solution protein hydrolysis method with superheating in a time shorter than 2 min. Protein sequence information was well recovered with the efficient and highly specific cleavage at Asp. Using nanoESI-MS, this method has been validated for the analysis of standard proteins such as ubiquitin. The application for phosphoprotein and glycoprotein analyses has also been explored. By incorporating a reducing agent into the protein solutions, disulfide bonds could also be cleaved along with fast hydrolysis, allowing for analysis of large proteins such as bovine serum albumin. The procedures for protein treatment and analysis were significantly simplified by using a high-temperature microreaction system. The integrated microreaction system is of low cost and requires low power; this can be important for further transferring it into POC applications. The potential of the microreaction system for biomarker analysis in clinical applications was also demonstrated using a miniature mass spectrometer for MS analysis, determining two antigens of Mycobacterium tuberculosis infection, a 10 kDa culture filtrate protein (CFP-10) and a 6 kDa early secretory antigenic target (ESAT-6).It is highly desirable to have fast and simple procedures developed for MS-based protein analysis, which would be important not only for proteomic research but also for applying protein analysis in clinical diagnosis.e.g. 150 °C) within 20 s  was loaded into the PFA tubing using a syringe; both ends of the tubing were then sealed by using two removable stoppers and the heating was performed for less than 2 min; after cooling in air for 30–60 s, the protein solution was transferred into a glass capillary (i.d. 0.8 mm) with a pulled tip in an offline fashion for subsequent nanoESI-MS analysis. For high-temperature reaction, although this method avoided applying high pressures on the protein aqueous solution, a high-pressure environment may still be created since a temperature over the boiling point of water at atmospheric pressure was applied . Fast and selective cleavage of aspartyl sites (X-Asp and Asp-X) of proteins or peptides could be achieved under the high-temperature and high-pressure conditions  and loss of NH3 from glutamine (Gln) at the N-terminal were commonly observed during hydrolysis of proteins.29 This was also observed in the high-temperature hydrolysis method, for example, #4 peptide  was observed at m/z 707.37 (2+), with a mass loss of 17.06 Da (–NH3). Using a Q-TOF instrument, the peptides can be identified by both the accurate mass and MS/MS experiments, as shown in Fig. S3.Hydrolysis of ubiquitin  was performed to evaluate the effectiveness and efficiency of the microreaction system. Ubiquitin aqueous solution  of 50 μL was prepared for demonstration, which was hydrolyzed at 150 °C for 2 min. Analysis was performed by nanoESI-MS in the positive ion mode on a quadrupole time-of-flight (Q-TOF) mass spectrometer. As shown in 650–1250 , suggest−1). Weak organic acids such as formic acid and acetic acid were reported to exhibit selective hydrolysis of proteins at the sites of X-Asp and Asp-X, while strong inorganic acids such as hydrochloric acid and sulfuric acid were often used for a more complete, but less selective hydrolysis of proteins.30,31 We compared the use of different types of acids, including formic acid, acetic acid, and hydrochloric acid. The pH values of the aqueous solutions were adjusted to 2.5, and the reaction was performed under 150 °C for 2 min. As shown in m/z 700.05), QQRLIFAGKQLE (m/z 707.37) and the +10 peak of ubiquitin (m/z 857.33), formic acid was found to have higher hydrolysis efficiency. Presumably this was due to a higher concentration of formic acid in aqueous solution of the same pH value, when comparing with acetic acid and hydrochloric acid. Since the dissociation of formic acid in the aqueous solution is an endothermic reaction, more H+ would be produced at high temperatures. This would benefit the hydrolysis reaction that is catalyzed by H+ in aqueous solution. In a further test on the effect by acid concentration, the efficiency of ubiquitin hydrolysis increased dramatically with the increase of formic acid contents from 0.02% to 2% (v/v)  . AlthougThe effect of temperature was investigated in the range of 60–180 °C. As shown in The peptide distribution of ubiquitin obtained by the high-temperature hydrolysis and nanoESI-MS was compared with the theoretical peptide distribution of ubiquitin through breakdown at the aspartyl site. As shown in Fig. S4,−1  was obtained for the analysis of ubiquitin. Besides nanoESI-MS, the fast hydrolysis method can also be coupled with LC-ESI-MS for protein analysis. For example, the peptides hydrolyzed from ubiquitin were well separated by reverse-phase LC and detected by high-resolution MS  and tris(2-carboxyethyl)phosphine (TCEP), which can break the disulfide bonds groups in proteins or peptides. The reduction is usually performed along with protease-based digestion and requires 0.5–1 h for incubation and reaction. In this work, an attempt was made to incorporate the reduction of disulfide bonds into the protein hydrolysis process.Linking through disulfide bonds (S–S) between two cysteine residues is a common type of post-translational modification in proteins during protein biosynthesis inside the cell.e.g., from +46 to +85) of the MS spectra of BSA in the positive ion mode (m/z 500–1200 (546.30–4872.49 Da), showing cleavage of proteins at aspartyl sites (m/z 500–1200 (546.30–6429.30 Da), while no obvious peaks of intact proteins were observed. This showed that complete cleavage of peptides of BSA was achieved by combination of separated procedures of reduction and hydrolysis. To prevent possible re-formation of disulfide bonds, 2-iodoacetamide  was added after reduction by DTT. As shown in Fig. S6,A series of experiments were performed for this investigation with bovine serum albumin , which has 17 disulfide bonds and 40 aspartyl sites. Treatment of BSA with a standard reduction process (12 mM DTT for 1 h under 50 °C) showed a significant charge shift , while no obvious peaks of intact proteins were observed  was introduced into the BSA solution  and then the mixture was treated using the microreaction system (150 °C). IAA was not used in order to simplify the sample preparation procedures. It was interesting that after reaction for 5 min, abundant peptide peaks were also detected in the range of observed . This reobserved  with TECobserved  was justN-glycosylated sites.7 Using solutions of HRP , relatively high hydrolysis efficiency was obtained by the high-temperature reaction, with the peaks of intact protein  decreased significantly after hydrolysis (m/z 300–1500 in the positive ion mode (https://www.uniprot.org/uniprot/P00433), 59% of the sequence can be identified , these peaks should be glycopeptides (Table S5N-acetylhexosamine (HexNAc), and pentose (Pent)  was used for demonstration, which consists of 308 amino acid units, 21 aspartyl sites and a number of drolysis . Highly ion mode . In compd Fig. S9. Besides Table S5 after cl Fig. S10.m/z 736.8 ([M + 2H]2+) with a high intensity. Due to the presence of Na+ and K+ in the phosphopeptide sample, peaks of m/z 747.8 ([M + H + Na]2+) and m/z 755.8 ([M + H + K]2+) were also obtained. After the high-temperature hydrolysis, several singly charged peaks were clearly observed in the range of m/z 1000–1300. The peak at m/z 1211.5 was identified as the peptide residual from cleavage of an aspartyl at the C-terminal and the phosphate group, while m/z 1006.5 was identified for cleavage of both aspartyl sites and the phosphate group . A solution of the phosphopeptide  was analyzed using the high-temperature hydrolysis followed by nanoESI-MS analysis. As shown in 35 For many protein biomarkers, their identification might only require the detection of presence of intact proteins, which can be realized by immunoaffinity methods. For many other protein biomarkers, however, the identification would require confirmation of certain peptide sequences for the purpose of accurate diagnosis of diseases. For example, CFP-10 and ESAT-6 are two biomarkers of Mycobacterium tuberculosis (Mtb) infections for the diagnosis of tuberculosis (TB) disease.36 The detection of intact CFP-10 and ESAT-6 using immunoaffinity methods can cause false positive results in TB diagnosis, since some nontuberculous mycobacteria can also secrete similar proteins.37 Comprehensively screening peptide sequences has revealed that some peptides of high Mtb specificity, such as the tryptic digests of CFP-10 (TDAATLAQEAGNFER) and ESAT-6 (WDATATELNNALQNLAR), can be used for accurate diagnosis of TB.38Fast determination of protein biomarkers is significant for clinical analysis and disease diagnosis.m/z 550–1100 in the positive ion mode. Four typical peptides from the hydrolysis of four aspartyl sites were well detected , m/z 602.37 , m/z 1018.24 , and m/z 888.43 . Good hydrolysis efficiency was also obtained for recombinant ESAT-6 , m/z 983.80 , and m/z 944.83 and m/z 1181.04 .In this study, we explored the fast analysis of peptides from CFP-10 and ESAT-6 by the high-temperature hydrolysis and nanoESI-MS method. Recombinant CFP-10 (MW: 11.73 kDa) and recombinant ESAT-6 (MW: 11.11 kDa) were used. CFP-10 in formic acid solution was detected with +8 to +13 charges by using nanoESI and Q-TOF . As showdetected  at m/z 8t ESAT-6 . Three tt ESAT-6  at m/z 1Mtb, we compared the sequences of CFP-10 and ESAT-6 with related proteins from a series of tuberculous and nontuberculous mycobacteria.36 As show in Mtb and M. bovis, while peptides #1 and #4 can also be found in nontuberculous mycobacteria such as M. kansasii, M. marinum and M. ulcerans.39 Although the sequence of the peptide #1 . These results showed that the high-temperature hydrolysis and nanoESI-MS method has the potential to be a useful tool for fast determination of protein biomarkers of TB disease.To confirm if the peptides obtained from high-selective hydrolysis were specific to 40,41 Previously we had developed several direct sampling methods and miniature MS systems for the fast analysis of drugs, metabolites and lipids.42–49 Recently, we also explored the application of miniature mass spectrometers in quantitation of synthetic peptides with molecular weights larger than 1500 Da.50 However, due to the sophistication of sample preparation procedures, fast analysis of proteins and digested peptides by miniature MS is still difficult.Miniaturization of the MS systems is an important component for implementing MS in clinical and POC applications.m/z 2000,51 which enabled detection of major peaks of small proteins such as ESAT-6 (m/z 1081) was relatively low, but the characteristic peptide  was detected at high abundance  MS system. It was coupled with a nanoESI source, where voltages of 1500–1800 V were used for analysis of proteins and peptides. This miniature MS system was capable of accurate MS analysis up to s ESAT-6 . In comps ESAT-6 , an obvis ESAT-6 . It probbundance . AlthougIn this work, a microreaction system was developed for the fast and direct analysis of proteins. Proteins were hydrolyzed in the microreactor under high-temperature (∼150 °C) and weakly acidic conditions, enabling selective breakdown of a series of proteins at the aspartyl site in 2 min. Being free of salts, the hydrolyzed solutions from the microreaction system can be directly used for nanoESI-MS analysis. By incorporation of reductants into the solution, one-step cleavage of peptide bonds and disulfide bonds was also realized, allowing for fast analysis of large proteins. The microreaction system is of small size, avoids using high pressures and only requires simple procedures for protein hydrolysis; the coupling of the microreaction system with a miniature mass spectrometer represents a useful tool for POC analysis of protein biomarkers.The authors declare no conflicts of interest.SC-011-D0SC03237A-s001


Muscle wasting is a symptom of the cancer cachexia closely related to the imbalance between protein synthesis and degradation. MyomiRs are small RNA molecules that do not encode proteins and have the function of regulating protein-coding genes, and in this way, myomiRs can regulate the homeostasis of skeletal muscle cells submitted to physiological or pathological stimulus. Aerobic exercise training (AET) is a nonpharmacological adjuvant treatment to prevent cancer cachexia, improving the patient’s quality of life. MyomiRs are modulated by cancer and AET, as well. Thus, we propose to investigate the effects promoted by AET on circulating and skeletal muscle myomiRs in cachectic and non-cachectic cancer mice. Exercise is a promising therapy for cancer-associated muscle wasting, revealing the importance to understand the molecular mechanisms involved to preserve muscle mass.We investigated the effects of AET on myomiRs expression in the skeletal muscle and serum of colon cachectic (CT26) and breast non-cachectic (MMTV-PyMT) cancer mice models. Colon cancer decreased microRNA-486 expression, increasing PTEN in tibialis anterior muscle (TA), decreasing the PI3K/mTOR protein pathway, body and muscle wasting, fibers’ cross-sectional area and muscle dysfunction, that were not preserved by AET. In contrast, breast cancer decreased those muscle functions, but were preserved by AET. In circulation, the downregulation of microRNA-486 and -206 in colon cancer, and the downregulation of microRNA-486 and upregulation of microRNA-206 expression in breast cancer might be good cancer serum biomarkers. Since the microRNA-206 is skeletal muscle specific, their expression was increased in the TA, serum and tumor in MMTV, suggesting a communication among these three compartments. The AET prevents these effects on microRNA-206, but not on microRNA-486 in MMTV. In conclusion, cancer induced a downregulation of microRNA-486 expression in TA and serum of CT26 and MMTV mice and these effects were not prevented by AET; however, to MMTV, the trained muscle function was preserved, probably sustained by the downregulation of microRNA-206 expression. Serum microRNA-206 is a potential biomarker for colon (decreased) and breast (increased) cancer to monitor the disease evolution and the effects promoted by the AET. Cachexia is a syndrome associated with several neoplastic diseases. Cancer cachexia is characterized by the progressive loss of lean body mass, with or without the loss of adipose tissue that can be partially but not totally reversed by diet support , and theCancer cachexia, one of the main manifestations of the damage that neoplastic diseases cause, causes muscle atrophy, which is the result of an imbalance between protein synthesis and degradation in skeletal muscle . The reaAccumulating evidence shows that the expression of microRNAs in skeletal muscle modifies cancer cachexia in experimental animals and humans . The micEvidence shows that exercise training provokes remarkable adaptations in skeletal muscle in healthy and pathological processes. One of the most marked effects is recovering the muscle protein balance ,13 that There is no evidence about the effects of AET on the expression of myomiRs in the cachectic and non-cachectic state associated with cancer. Therefore, our goals are to investigate the profile of myomiRs expressed in CT26 colon cancer cachectic and MMTV-PyMT mammary cancer non-cachectic mice. In addition, to investigate the effects of AET on the myomiRs and skeletal muscle phenotype and function, as well as evaluate whether cancer cachexia and AET are modulating the expression of circulating myomiRs.6 of CT26 colon cancer cells . These mice were randomly assigned in colon cancer sedentary  and colon cancer trained  groups. A control group was also assigned in wild type sedentary  and wild type trained  groups. MMTV  c57 female mice that represent non-cachectic transgenic mice with spontaneous tumor appearance were also studied [n = 8) and mammary cancer trained  groups. A control group was assigned in wild type sedentary  and wild type trained  groups. The mice were provided from the Medical School of the University of Sao Paulo animal facility. The animals were housed 5 per cage at controlled room temperature (22 °C) with a 12-h dark–light cycle and were fed standard mouse chow and had access to water ad libitum.Balb/c female mice that represent a cachectic model were injected with 1 × 10 studied . They weThe training protocol was performed on a treadmill according to the protocol developed by Ferreira et al. . Mice weExercise capacity was assessed by maximal distance running in the maximal exercise test. The exercise test protocol was conducted, as described by Ferreira et al. . BrieflyThe grip test was used to evaluate limb traction force and muscular function in the CT26 model. The mice were positioned in the equipment , suspended by the tail, and stimulated to draw the grid coupled to a dynamometer. Three measurements were performed, and the highest value was selected. The grip test was performed at the end of the training protocol.The ambulation behavioral test was used for the analysis of skeletal muscle function in the MMTV-PyMT model. This test estimates the contractile force and motor coordination. Mice hind legs were placed in contact with non-toxic black paint and then placed inside a rectangular wooden box of 1 m (homemade and lined with white paper) for walking. Subsequently, the stride length was measured.After euthanasia, the soleus, tibialis anterior (TA), gastrocnemius and plantaris muscles and tibia bone were harvested and weighed. Muscle mass was assessed by the measurement of the ratio of tissue weight in grams to tibia length in millimeters (muscle weight g/mm).g for 15 min at 4 °C, and the supernatant was used to perform the enzymatic kinetics. Protein was measured using the method of Bradford et al. [v/v) Triton X-100 and the homogenate. The reaction was initiated by the addition of 18.9 mM oxaloacetate to the reaction mixture. The enzymatic kinetics was recording and performed at 25 °C for an interval of 10 min, at 412 nm using Victor . The result of the enzyme activity was expressed in nmol·min−1·mg protein−1.To evaluate citrate synthase activity, the gastrocnemius muscle was homogenized in 1 g tissue: 7 µL of extraction buffer containing 50 mM Tris-base, pH 7.4 plus 1.0 mM EDTA at 4 °C. The samples were centrifuged at 3000× d et al. , using bd et al.  from the−ΔΔCT. Results were expressed as % of control groups (WT).Frozen TA skeletal muscle samples were used for RNA extraction. Fifteen milligrams of the tissues were homogenized in Trizol (700 μL), and RNA was isolated according to the manufacturer’s instructions . Serum RNA was extracted through the miRNeasy Serum/Plasma Kit  following the manufacturer’s instructions. MicroRNAs expression was measured using real-time PCR and the TaqMan MicroRNA Assay  as specified by the manufacturer’s instructions. A detailed method was already described by Fernandes et al. [® O.C.T, frozen in isopentane and then in liquid nitrogen. Immunohistochemistry was conducted to analyze the cross-section area; sections (10-micrometer thick) were obtained from TA muscle using a Leica-CM 1850 cryostat . The muscle sections were fixed with 4% formalin  for 10 min at room temperature, permeabilized in 0.2% Triton X-100  and 1% bovine serum albumin  diluted in phosphate buffer saline  for 10 min. Blocking was performed with 10% goat serum  in PBS for 45 min. Glass slides were incubated with a solution containing the primary antibody anti-laminin  for delimiting muscle fibers, with 1.5% goat serum in PBS for 1 h 30 min at room temperature. After proper washing, the sections were incubated with a solution with a secondary fluorescent antibody . The images were captured with a 200× magnification and a 20× objective. The images were recorded on a computer connected to a fluorescent microscope and connected to a photographic system  . The quantification of the cross-sectional area for each fiber was evaluated using the program Image J software . The results were expressed in μm2.TA skeletal muscle was harvested and embedded in Tissue-TekThe protein levels PTEN, PI3K, AKT, mTOR, FOXO3a and FOXO3a phosphorylated (pFOXO3a) in the TA muscle were analyzed using Western blotting. Samples were loaded to SDS-PAGE on polyacrylamide gels (6–15%). After electrophoresis, proteins were electro-transferred to a nitrocellulose membrane . Equal loading of samples (30μg) and transfer efficiency were monitored with the use of 0.5% Ponceau S staining of the blot membrane. The blot membranes were incubated with polyclonal antibodies anti-AKT , anti-phospho-AKT-SER473 , anti-PI3K , anti-PTEN , anti-mTOR , FOXO3a , FOXO3ap  and anti-GAPDH . The bands were analyzed using Image J software . Skeletal muscle GAPDH expression levels were used to normalize the results, which are expressed as a percentage of control expression.p ≤ 0.05 were accepted as statistically significant. The Tukey post hoc test  was used for individual comparisons between means when a significant change was observed with ANOVA.The results are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA. To indicate how closely two variables, change in relationship to each other, Pearson’s correlation coefficient was used. Probability values of p > 0.05 , fourth week (4WK-AET) and end of the study (POST-AET) A,B. The p > 0.05 B.p = 0.02), WT POST-AET , CT26 POST-AET  and CT26+TR POST-AET .The maximal exercise capacity significantly increased in the WT+TR group at POST-AET (1021 m ± 121.8) compared with PRE-AET  and WT group POST-AET . The exercise capacity in the CT26+TR group POST-AET (470.5 m ± 16.7) significantly decreased compared with CT26+TR 4wk-AET  and WT POST-AET , but not compared with CT26+TR PRE-AET   compared with the PRE-AET ( > 0.05) B.−1·mg protein−1) increased by 79% in the WT+TR  group compared with WT (3.5 ± 1.7), while it was decreased in the CT26  and CT26+TR  groups compared with WT+TR. There were no changes among the WT, CT26 and CT26+TR groups  and CT26+TR POST-AET  groups were decreased at the end of the training protocol period compared with the WT POST-AET (25.2 ± 0.6) group. Additionally, the tumor-free body mass was decreased in CT26 POST-AET compared with CT26 PRE-AET   D.Altogether, these findings showed that CT26 cachectic mice display exercise intolerance, paralleled by lower body mass and oxidative metabolism, which were not prevented by AET.p > 0.05 , fourth week (4WK-AET) and end of the study (POST-AET) A,B. The p > 0.05 B.p = 0.04) and MMTV POST-AET , MMTV PRE-AET (p = 0.04) and MMTV 4k AET (p = 0.03). In the MMTV group, physical capacity significantly decreased at POST-AET compared with PRE-AET , WT POST-AET (p = 0.04) and MMTV+TR POST-AET . The MMTV+TR group showed no significant change in exercise capacity compared with WT POST-AET  compared with WT POST-AET  was increased by 33% in the WT+TR (3.6 ± 0.9) group compared with the WT  group and 39% compared with the MMTV  group. AET restored the enzyme activity in the MMTV+TR (3.0 ± 0.8) group to the same levels of the WT+TR group  and CT26+TR  groups showed muscle wasting compared with the WT (2.02 g/mm ± 0.1) group. Similar results were observed in the gastrocnemius muscle where the CT26  and CT26+TR  groups showed muscle wasting compared with the WT (6.42 g/mm ± 0.2) group. There was no effect of AET in restoring the muscle wasting of the TA and gastrocnemius  decreased in the CT26 group and 28% (p = 0.01) in CT26+TR compared with WT. Therefore, there was no effect of AET (The grip strength was 41% (t of AET B.2) of the muscle fibers was smaller in the CT26  and CT26+TR  groups compared with the WT (2879.4 ± 359) and WT+TR (2870.5 ± 345) groups (2) represented by the total fibers number percentage.The cross-sectional area (µm) groups C,E. FiguTA muscles were also evaluated in the MMTV-PyMT non-cachectic mice. There were no significant changes in muscle mass in the MMTV-PyMT non-cachectic mice regardless of exercise training A. The MMThe expression of microRNAs-1, -133a, -133b, -206 and -486 were evaluated in the TA muscle of both cancer models, CT26 cachectic and MMTV-PyMT non-cachectic mice. In addition, microRNAs-206 and -486 expression was evaluated in the mice serum from both models. No significant differences in myomiRs-1, -133a and -133b expression were found among groups in both cancer models .p = 0.03) and CT26+TR  groups compared with the WT (100%) group. No significant difference was found between the WT+TR and WT groups (p = 0.01), CT26  and CT26+TR  groups compared with WT (100%) (p = 0.03), MMTV  and MMTV+TR  groups compared with WT (100%) (p = 0.002) and MMTV+TR  groups compared with WT (100%). No significant difference was found between the WT+TR and WT groups  C. In theT (100%) B. The seT groups D.p = 0.04) group compared with the WT (100%) (p = 0.02), WT+TR  and MMTV+TR  groups, while it was decreased in the WT+TR  group compared with the WT group (p = 0.0003), WT+TR  and MMTV+TR  groups  C group. WT group B. The se) groups D.p = 0.02) group compared with the MMTV group  and CT26+TR  groups compared with the WT (100%) group  and CT26+TR  groups compared with the WT (100%) group and also it was decreased in the CT26  and CT26+TR  groups compared with the WT+TR group (p = 0.008) and CT26+TR  groups compared with the WT+TR group  group A, and it%) group A. PI3K pTR group B. LikewiTR group D. In addp = 0.05) and MMTV+TR  groups compared with the WT+TR (100%) group (p = 0.05) and MMTV+TR  groups compared with the WT+TR group, although there was no change in pAKT expression among the groups  group B. In thee groups C. In adde groups D,E.We showed that cancer induced a downregulation of microRNA-486 expression in the TA skeletal muscle and serum of CT26 and MMTV mice. These effects were not prevented by AET. Furthermore, cancer induced a downregulation of microRNA-206 expression in the serum of CT26 mice. Additionally, microRNA-206 was upregulated in the TA skeletal muscle, serum and tumor of the MMTV model, and these effects were prevented by AET. In addition, circulating myomiRs have been suggested as a prognosis cancer biomarker, thus the downregulation of miRNA-486 and -206 in colon cancer, as well as the downregulation miRNA-486 and upregulation of miRNA-206 in breast cancer might be good serum biomarkers to these two kinds of cancer. In addition, the serum miRNA-206 expression of CT26 and MMTV can be a good biomarker of the effects of AET preventing the effects of cancer, as well as of miRNA-206 in TA. According to our knowledge, this is the first study showing the characterization of the myomiRs’ expression in CT26 and MMTV-PyMT animal models submitted to an AET protocol.The main findings observed in the CT26 model were microRNA-486 expression downregulation in TA skeletal muscle in CT26 and CT26+TR groups, increased protein PTEN levels decreasing the PI3K/Akt/mTOR pathway and AET not being effective in restoring the crucial protein synthesis pathway in skeletal muscle.We are the first to show the possible role of microRNA-486 in CT26 mice skeletal muscle. Our results suggest that the decreased expression of microRNA-486 leads to a loss in skeletal muscle mass, function and aerobic capacity since it causes impairment in the protein synthesis pathway by targeting the PTEN pathway. These data corroborate a study by Small et al.  that invThe decreased expression of microRNA-486 in cancer groups of the MMTV-PyMT model were not followed by changes in PTEN protein expression. However, PI3K and total AKT (not pAKT/AKT) protein expression were downregulated in MMTV and MMTV+TR compared with the WT group. Chen et al.  also invThe microRNA-206 expression was upregulated in the MMTV group and downregulated as an effect of AET in WT+TR and MMTV+TR B. SkeletTherefore, our results suggest that the downregulation of microRNA-486 and the upregulation of microRNA-206 are contributing to the impairment in exercise capacity and muscle functioning in the MMTV group that is prevented by AET with the downregulating of microRNA-206 expression in the MMTV+TR group. This result is probably due to the fact that microRNA-206 is a skeletal-muscle-specific microRNA inducing more robust effects than a non-muscle specific microRNA.MyomiRs, in turn, are a class of microRNAs with enriched expression in skeletal and cardiac muscles that act by controlling myogenesis, homeostasis, muscle metabolism and regeneration . In termCT26 and MMTV sedentary mice showed low aerobic capacity, which was preserved by the AET B. These Circulating microRNAs promote cell to cell and tissue to tissue communication in an autocrine, paracrine and endocrine manner. The serum microRNA-486 expression was downregulated in the CT26, CT26+TR, MMTV and MMTV+TR groups C,D compaIn the CT26 cancer colon mice, the serum microRNA-206 expression in the CT26 group was downregulated compared to all the other groups C. Liu etIn the MMTV group, the TA and serum microRNA-206 expression was significantly increased compared with the other groups B,D. ThesFor a long time, microRNA-206 has been reported as having a tumor suppressor role in breast cancer . Once thIt is noteworthy that the results related to microRNA-206 expression in breast cancer and the effects of AET are unclearly established, sometimes they are contradictory, mainly regarding the effects of AET. MicroRNA-206 expression is linked to many malignancies and plays an important role in several cancers’ tissues ,39. In bIn conclusion, our results suggest mechanisms by which myomiRs can regulate skeletal muscle mass and function in colon and breast cancer. They also provide evidence for the effects of the AET on circulating myomiRs. CT26  mice, microRNA-486 downregulation results in TA and gastrocnemius cachexia and muscle dysfunction. These effects were not prevented by AET. Our findings showed, for the first time, the microRNA-486 expression in skeletal muscle cachexia resulting from colon cancer in mice. These results suggest that treatment with mimic-microRNA-486 may contribute to preserving the skeletal muscle mass and individual quality of life. The MMTV-PyMT  mice, downregulation of microRNA-486 expression and upregulation microRNA-206 in the MMTV cancer group corroborate the impairment in the skeletal muscle performance, although the muscle mass is still preserved. In contrast, AET decreases microRNA-206 expression in the MMTV model and improves aerobic capacity. These results suggest that microRNA-486 downregulation in the MMTV group can be the first signaling and early biomarker of muscle dysfunction, since it is also downregulated in the cachetic model, also suggesting that the effects of the microRNA-muscle-specific are more prevalent than a non-muscle-specific microRNA.Circulating microRNA-486 expression levels were downregulated in the sedentary and trained colon and breast cancer mice. Similar results were observed in the TA skeletal muscle, suggesting a link between the effects promoted by the tumor on skeletal muscle and circulating of microRNA-486 levels. The circulating microRNA-206 expression levels were very low in CT26 cancer mice, suggesting microRNA-206 as an independent prognostic indicator for colorectal cancer. AET increased the expression in both trained groups C. On theCT26 and MMTV mice have low aerobic capacity, which is a strong predictor of poor prognosis. More importantly, this change can be preserved by AET. Thus, AET might be an important non-pharmacological strategy for the treatment of patients with breast and colon cancer. MicroRNAs have the potential to be cancer biomarkers that can be modulated by AET. This is a promising area for future studies.


A key factor behind the unnecessary use of antibiotics is the lack of rapid and accurate diagnostic tests. In this study, we developed a novel and fast flow cytometric single-tube method to detect bacterial infections within 30 minutes.Quantitative flow cytometric four-colour analysis of host biomarkers CD35, CD64, CD329, and MHC class I expression on neutrophils and lymphocytes was performed on samples taken from 841 febrile patients with suspected infection. Obtained data was incorporated into the four-colour bacterial infection (FCBI)-index, using the developed bacterial infection algorithm.In distinguishing between microbiologically confirmed bacterial (n = 193) and viral (n = 291) infections, the FCBI-index method was superior to serum C-reactive protein (CRP) and procalcitonin (PCT). In 269 confirmed viral respiratory tract infections, 43% (95% CI: 37–49%) of the patients had an increased FCBI-index, suggesting probable bacterial coinfection.The proposed FCBI-index test might be a potent additional tool when assessing appropriateness of empiric antibiotic treatment.This study has been financially supported by Turku University Hospital  and The Finnish Medical Foundation. There is an ongoing need for new sensitive and specific markers of bacterial infection. One candidate is the flow cytometric determination of host biomarkers, i.e. receptors, on the surface of blood leukocytes. The hypothesis behind the idea is that bacterial and viral infections induce different systemic profiles of proinflammatory cytokines, which can lead to different expression patterns of certain cell surface receptors on blood leukocytes in these different infection types. This study is a continuation of our previous works on this novel and narrow field of research.As a result of persistent development work during the last two decades, we have now developed a novel and simple flow cytometry-based bacterial infection marker, the four-colour bacterial infection (FCBI)-index, which incorporates the quantitative analysis of CD35, CD64 and CD329 receptors on neutrophils and lymphocytes and detects febrile bacterial infections reliably in less than 30 minutes. Data of this comparative study clearly shows that the FCBI-index method is superior to CRP and PCT when distinguishing between bacterial and viral infections.The FCBI-index might be a potent part of antimicrobial stewardship measures in hospitals and health centers, helping physicians to decide whether empiric antibiotic treatment is necessary or not.Alt-text: Unlabelled box1A key factor behind the unnecessary use of antibiotics and the increasing global burden of antimicrobial resistance is the lack of rapid and accurate diagnostic tests Until now, the increased expression of Fc-gamma-receptor I (FcγRI/CD64) on neutrophils has been the most widely used (and only) commercially available flow cytometric marker of infection , as well as severity of sepsis Compared with neutrophil CD64, neutrophil complement receptor 1 (CR1/CD35) seems to be a more specific bacterial infection marker. In 2006, we discovered that the average expression level of CD35 on neutrophils in bacterial infections was over three-fold higher than in viral infections and healthy controls, displaying 85% sensitivity and specificity when distinguishing between bacterial and viral infections Since 2006, we have developed three more (flow cytometric) multiparametric score/index methods to distinguish between bacterial and viral infections: Bacterial Infection Score (BIS), which was the first pure flow cytometric bacterial infection marker, Bacterial Infection (BI)-index, and Two-Colour Bacterial Infection (TC-BI)-index Albeit highly sensitive and specific, a common drawback of the above-mentioned early multiparametric methods is their complexity. For example, the receptor analyses were conducted using isolated leukocytes, widening the time window from procuring blood samples to data handling and index/score-based diagnosis up to 45 min. In addition, one- or two-colour flow cytometric receptor analyses were performed in 2-3 separate test tubes. The lack of the universal leukocyte marker was still one more problem in these early score/index methods.Based on what we have learned from our previous works in this field, we can conclude that there are three basic principles underlying the concept of an efficient flow cytometric bacterial infection marker: It should be 1) multiparametric, 2) fast and practical with the minimum amount of sample handling, and 3) require only one measurement from a single test tube. Consequently, a novel state-of-the-art bacterial infection marker fulfilling all of the above criteria has now been developed. We present in this study the truly workable multiparametric flow cytometry method, the Four-Colour Bacterial Infection (FCBI)-index, which is based on detection of the relative number of CD35, CD64 and CD329 receptors on neutrophils and lymphocytes.In order to reduce the total sample handling time from over 45 min (above-mentioned previous methods) to less than 30 min in the present study, the incubation of the whole blood sample (instead of isolated leukocytes) with four fluorescent labelled receptor specific monoclonal antibodies, red blood cell lysis, and the flow cytometric run were performed consecutively in the same test tube without any time-consuming washing steps. In the data handling process, the conversion of the raw receptor expression data into a single FCBI-index value was performed using the novel bacterial infection algorithm.Of the studied inflammatory receptors, CD35 is expressed on granulocytes, monocytes, B cells, follicular dendritic cells, erythrocytes, NK cells and at low levels on T cells CD64 is expressed on monocytes, macrophages, circulating dendritic cells, and at low levels on resting neutrophils and lymphocytes Sialic acid-binding Ig-like lectin 9 (Siglec9/CD329) is a member of the sialic acid-recognition protein family that is highly expressed on neutrophils and monocytes, and only weakly on lymphocytes and NK cells Major histocompatibility complex I (MHCI) molecules, which bind endogenous  antigen peptides and present them to the CD8-positive cytotoxic T-cells ,23, are In this comparative study, we present the distribution of FCBI-index, CRP and PCT values primarily in bacteremia, microbiologically confirmed local bacterial infection, clinically diagnosed probable bacterial infection, and microbiologically confirmed viral respiratory tract infection (RTI).22.1The study protocol was approved by the Ethical Committee of Turku University Hospital district (Reference number: ETMK 23.11.2007 § 99). All research was performed in accordance with relevant guidelines and regulations. The study period covered four winter influenza seasons (October to March) from 2015 to 2019.Adult patients (aged 18 years or over) with a suspected infection were eligible for the study if they had a body temperature of at least 37.5°C. Patients with ongoing cytostatic treatment for a malignancy or biological treatment against autoimmune diseases such as rheumatoid arthritis or an inflammatory bowel disease were excluded, as well as patients with hematological malignancies with possible influence on white blood cells, and those who were hospitalized for over 48 h before procuring blood samples.Informed consent was obtained from all participants after the nature and possible consequences of the study were explained, both in writing and orally. The patient volunteers, as well as healthy controls, signed an approval form to give a further 3 mL lithium heparin blood sample for the receptor study. The sample was taken at the same time as the routine tests.The clinical patient data were collected from the confidential patient records by authorized clinician. The patients had the option to withdraw at any time during the study.The patients were treated according to normal clinical policy and the receptor analysis  had no influence on the treatment decisions.2.2For the purpose of this study, we attempted to confirm all diagnoses either microbiologically or serologically. It should be noted that the used methods were chosen on a clinical basis, so we did not have the opportunity to study all pathogens systematically.Bacterial culture served as a gold standard for the diagnosis of confirmed bacterial infections (n = 193). Confirmed bacterial infections included cases where bacterial culture from sources such as blood, urine, stool or needle aspiration from an abscess or empyema was positive. In addition to bacterial culturing methods, a positive bacterial antigen test was also accepted , if the clinical assessment supported the finding. A concomitant viral infection was diagnosed in 5.7% (11/193) of cases in this group .Confirmed viral infections (n = 291) were diagnosed using serological tests, PCR tests, or antigen detection tests. Confirmed viral infection required detection of IgM antibodies or an at least 4-fold increase in IgG antibody titer in the serum. At the start of the study, influenza was mainly diagnosed using the antigen detection test, but later, PCR tests became the main method for diagnosis of infection with influenza virus or other respiratory viruses. A concomitant asymptomatic bacteriuria (negligible bacteria level in urine) was diagnosed in 5.2% (15/291) of cases in this group .In addition, we also accepted some clinical diagnoses without definitive microbiological test results. Clinically diagnosed probable bacterial (n = 82) or viral (n = 21) infections were classified by an infectious disease specialist and were mainly cases of pneumonia and erysipelas infections of skin . PatientThe second biggest group of the study was composed of the patients with fever of other or unknown origin (n = 254). This heterogeneous group included patients with various diseases, such as pneumonia with unknown etiology , inflammatory diseases and cancer .At least two clinicians, one of which was an infectious disease specialist, made the final diagnosis.Note that the FCBI-index test was not used for making a clinical diagnosis.Disease-specific bacterial and viral pathogens are listed in Supplementary Tables 1 and 2, respectively.Group-specific antimicrobial prescriptions are listed in Supplementary Table 3.2.39/L) of leukocytes were determined using a Sysmex XE-2100 automatic blood cell analyzer . Measurement of the serum CRP level (mg/L) was based on a whole-blood immunoturbidimetric method. An electrochemiluminescent immunoassay (ECLIA) method was used to determine serum procalcitonin (PCT) level (ng/L). All tests, including flow cytometric receptor analysis, were performed using blood samples taken at the same time.Demographic and clinical characteristics of the patients at baseline are provided in Supplementary Table 4. Routine laboratory tests  were performed at the clinical laboratory (TYKSlab) of the Turku University Central Hospital. Total and differential counts : Two CyFlow Cube 8  flow cytometers with slightly different measuring capacities were used for the quantitative receptor analysis of blood leukocytes. One instrument (FCM1), located in the Department of Life Technologies, University of Turku, was used to analyse blood samples taken from patients treated at different medical wards of Turku University Hospital (n = 561). The other instrument (FCM2), located in the emergency room of Turku University Hospital, was used to analyse only those blood samples taken from emergency room patients (n = 280).Fluorescence-conjugated receptor-specific monoclonal antibodies (test kit): Phycoerythrin (PE)-conjugated anti-human CD35 (Clone E11) was purchased from Ancell (ANC-184-050), PerCP-Vio700-conjugated anti-human CD64 (Clone 10.1.1) was purchased from Miltenyi Biotec (130-101-422), fluorescein isothiocyanate (FITC)-conjugated anti-human CD329 (Clone 191240) was purchased from R&D systems (FAB1139F) and AF647-conjugated anti-human MHC class I molecule (Clone W6/32) was purchased from R&D systems (FAB7098R). (Supplementary Table 5).4Cl to the blood-antibody suspension and incubating for 5 min at room temperature, after which the run volume was adjusted to 1 mL by adding 650 μL of cold 1.54-fold calcium- and magnesium-free HBSS -buffer supplemented with 1.54 mg/mL gelatin (1.54 × gCMF-HBSS)  in saline, were mixed in one polystyrene flow cytometer vial and incubated for 15 min at room temperature. Red blood cell lysis was performed by adding 314 μL of 0.83% NHMF-HBSS) a.Fig. 2AGate settings in flow cytometric run: In the first phase of the flow cytometry run, the fluorescent leukocyte population within gate R1 was gated out from the SSC log/anti-MHCI log bivariate histogram (unfluorescent debris was excluded) b. In theFlow cytometric data analysis: For the data analysis, three additional bivariate histograms, namely SSC/CD35, SSC/CD64, and SSC/CD329 were made, showing mean fluorescence intensities  of gated neutrophil (R2) and lymphocyte (R3) populations c. In theCalculation of FCBI-index value: The FCBI-index value was simply calculated by substituting the obtained receptor-specific MFI data from neutrophil and lymphocyte populations into the bacterial infection algorithm d:FCBI−inConstant denominator  and power  values in the algorithm were obtained by iteration  in order to achieve the best differentiation between bacterial and viral infections by the final FCBI-index . In order to achieve the wide dynamic range of eight decades of the method, all three variables must be incorporated into the FCBI-index. Neutrophil CD35 is the cornerstone variable of the FCBI-index, whose differential capacity is supported by the other two variables, CD64 and CD329. A more detailed step-by-step description of the development of the bacterial infection algorithm is presented in the Supplementary Figs. 1-5.2.6Reproducibility of the FCBI-index method was tested in two different conditions. First, we tested on how gate settings during flow cytometric data analysis b and 2c According to the presented evidence, it can be concluded that the reproducibility of the FCBI-index method is good.2.7Sixty patient samples were excluded because of deficiencies in the quantitative receptor analysis . We anal2.8The following formula was used to estimate necessary sample size for reliable differentiation between bacterial and viral infections:2 × StdDev × (1-StdDev)]/(Margin of error)2Necessary sample size (n) = [(Z-score)2 × 0.5 × 0.5/0.052 = 384 samplesIn calculations, we chose a 95% confidence level (Z-score = 1.96), 0.5 standard deviation and a margin of error  of +/- 5%: n = 1.96However, from our earlier similar cohort studies we have learned that the proportion of the patients with confirmed diagnosis of infection  from all febrile patients with suspected infection is about 40%,12. Ther2.9This study has been financially supported by Turku University Hospital  and The Finnish Medical Foundation (grant 4084/2014).The funding sources played no role in the study design, data collection, data analysis, interpretation, writing of the report, and the decision of paper submission.3In this comparative clinical study, we developed, evaluated and applied a novel flow cytometric method, a mathematical algorithm called the FCBI-index . Three mUsed flow cytometry (FCM1 or FCM2) did not have statistically significant influence on the final FCBI-index distributions .Neither age nor gender have any influence on the detection of bacterial infection using the FCBI-index .3.1There were two main observations supporting the applicability of the FCBI-index as a potent bacterial infection marker. Firstly, the area under the receiver operating characteristic (ROC) curve (AUC) statistic showed that the FCBI-index had an AUC of 0.901 (95% CI: 0.874–0.927), superior to that of CRP  and PCT  in distinguishing between microbiologically confirmed bacterial (n = 193) and viral (n = 291) infections .Of 193 patients with microbiologically confirmed bacterial infections, 11 also had concomitant confirmed viral infection, of which 10 (91%) had an FCBI-index ≥ 1.0 , indicating that simultaneous viral infection does not have a significant influence on the detection of bacterial infection using the FCBI-index.Since there were not statistically significant differences in the pairwise comparisons of FCBI-index, CRP and PCT values between bacteremic (n = 26), microbiologically confirmed local (n = 13), and clinically diagnosed probable (n = 32) bacterial pneumonia subgroups [p-valuesPairwise between group comparisons showed that the FCBI-index and CRP values of the bacterial pneumonia subgroup were statistically significantly increased when compared with the six virally infected RTI subgroups and patients having pneumonia with unknown etiology  in 97% (95% CI: 93-100%) of the patients with bacterial pneumonia and in 87% (95% CI: 79-100%) of the patients with pneumonia with unknown etiology, while the corresponding percentages were 93% (95% CI: 87-99%) and 79% (95% CI: 70-100%) for CRP, and 79% (95% CI: 70-100%) and 44% (95% CI: 32-56%) for PCT, respectively. On the other hand, an increased PCT value was observed in 93% (95% CI: 83-100%) of the patients with pneumonic influenza B infection and in 46% (95% CI: 36-56%) of the patients with non-pneumonic influenza A infection, while the corresponding percentages were 65% (95% CI: 47-83%) and 21% (95% CI: 13-29%) for CRP, and 54% (95% CI: 35-73%) and 17% (95% CI: 10-24%) for the FCBI-index, respectively. According to the Pearson chi-square analysis, the above-mentioned differences in the percentages of patients with increased parameter values between the three compared methods were significantly different .3.2The positive effect of the wide dynamic range of the method on bacterial-viral differential diagnostics could be seen when the minimum value of the bacterial pneumonia group was used as an alternative cutoff value  in patients with RTI .With the FCBI-index method, possessing the widest dynamic range of eight decades, 43%  of the patients with viral RTI had an FCBI-index ≥ 0.36, indicating the possibility of bacterial coinfection. This percentage is in line with the meta-analysis of 27 prospective studies by Klein et al., showing that between 2% and 65% of the patients with laboratory-confirmed influenza had a bacterial coinfection In the case of PCT and CRP, which have a dynamic range of four and three decades, the use of the alternative cutoff values of 28 ng/L and 14 mg/L led to the suspiciously high proportion of probable bacterial coinfection: 98% and 83%, respectively.Of those 16 virally infected patients with clinical signs of bacterial pneumonia (lobar pneumonia detected by chest radiograph or pneumonia with CRP higher than 200 mg/L), 94% (15/16) had an increased (≥ 0.36) FCBI-index value .3.3In retrospect, the prevalence of symptom-based empiric antibiotic prescription for the patients with febrile viral RTI was 69%  in this study a.Of the 185 antibiotic-treated patients, only 52%  was 77% . Puumala virus infection was a special case among the other viral infections, in that all 10 patients suffering from it had an increased PCT value  and seven also had an increased FCBI-index value , both reflecting the severe clinical symptoms of this viral infection. Ninety percent of the Puumala virus-infected patients were treated with empiric antibiotics before the diagnosis was confirmed.4Clinicians are often tempted to prescribe symptom-based antibiotics “just to be on the safe side” to avoid the onset of a severe and possibly life-threatening bacterial infection We have developed a novel and simple flow cytometric-based bacterial infection marker, the FCBI-index, to detect febrile bacterial infections reliably within 30 minutes. The method is based on the quantitative analysis of host markers CD35, CD64, and CD329 receptors on neutrophils and lymphocytes. The fourth member of the antibody kit, an anti-human MHCI antibody, was exploited as a pan-leukocyte marker in order to sort fluorescent leukocytes from non-fluorescent debris. The reagent cost per test, consisting mainly of presented antibody kit, is only about 5.5 € (6.6 $).The incubation of the whole blood sample with the receptor-specific antibodies, red blood cell lysis, and flow cytometric run were carried out one after another in the same test tube without any time-consuming washing steps. This differs from our previous studies, where antibody incubation was performed with isolated leukocytes. The FCBI-index test requires only a minimal amount of the whole blood sample (22 μL), making it possible to utilize a fingertip blood sample as the starting material In the data handling process, the incorporation and conversion of the raw receptor expression data into a single FCBI-index value was performed using the mathematical bacterial infection algorithm. In this study, the FCBI-index value was calculated by substituting the obtained receptor-specific MFI data into the bacterial infection algorithm manually, but it should be relatively easy to create flow cytometry data analysis software for automated FCBI-index calculation.An incomplete screening of the bacterial pathogens among virally infected febrile patients, as well as the lack of day-to-day follow-up of how the FCBI-index values vary during the course of the infection, were the most noteworthy study design-related limitations of the presented research. We plan to conduct a future clinical follow-up study addressing the above issues with a limited patient cohort. In particular, patients diagnosed with inflammatory conditions , whose clinical presentation and FCBI-index distribution seemed to be quite similar to that of bacterial infection , as well as immunocompromised patients will be a subject of special attention when selecting control groups for these future studies. Another limitation of the method itself is that FCBI-index-based determination of the exact type of bacterium (or virus) causing the infection is impossible. Therefore, the use of the FCBI-index method as a preliminary test preceding and directing: a) confirmatory identification of pathogens via PCR, antigen detection or enzyme-linked immunosorbent assay (ELISA) tests, and b) empiric antibiotic treatment, for example, would be the best strategy to achieve optimal economic and clinical benefits.Of course, several studies of independent research groups will be needed in the future to confirm our observation that the used flow cytometry does not have significant influence on FCBI-index based distinguishing between bacterial and viral infections. When calculating FCBI-index values in future clinical studies, we recommend primarily using predetermined constant denominator  and power  values in the algorithm. However, if one wants to make some changes to these constant values, they must be minor in scale and should be performed gradually. For example, if the power value of 3.1 is used instead of 3, then the final FCBI-index value will be about 0.9-1.3-fold compared with an original one, depending on CD35 RATIO value of course. On the other hand, if the denominator of 3.5 is used instead of 3.6 when calculating CD35 RATIO, then the final FCBI-index will be 1.088-fold compared with an original one.Based on the presented evidence, we can speculate that the FCBI-index value in combination with the clinical course of the febrile illness can be utilized to give reliable recommendations for or against the initiation of empiric antibiotic therapy. From an ethical standpoint, the risk of a life-threatening bacterial infection should always be eliminated. Therefore, the routine use of a single FCBI cutoff value does not necessarily lead to the best balance between cost-effectiveness and treatment outcome, the latter being the first priority. Instead, it may be more beneficial to employ three FCBI cutoffs to ascertain the appropriate antibiotic prescription using the FCBI-index a. Two cuRetrospectively, we can speculate that, if the antibiotic treatment-related course of actions described above had been performed in the patients with RTI symptoms in this study, then an immediate empiric antibiotic would have been prescribed for 115 (43%) of the 269 patients with a viral RTI, 38% less than the actual 185 prescriptions given.We conclude that the proposed FCBI-index method, particularly when associated with inexpensive, portable and easy-to-use flow cytometer, might be a potent part of antimicrobial stewardship measures in hospitals and health centers, helping physicians to decide whether empiric antibiotic treatment is necessary or not.J.N. and P.J-K. contributed to project conceptualisation and study design. J.N. contributed to flow cytometric methodology and literature search, J.N., U.H. and P.J-K. contributed to sample collection and investigation of samples, P.J-K. and U.H. contributed to administration of patient records, J.N. and P.J-K. contributed to data analysis and data visualisation. J.N. J.O. and P.J-K. provided project supervision. All authors contributed to writing and editing. All authors read and approved the final version of the manuscript. All authors have verified the underlying data.The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information file. Extra data are available from the corresponding author upon request (jarnuu@utu.fi).P.J-K declares consulting fees from MSD and GSK, participation on an advisory board for MSD and GSK, and stock in Orion, all outside of the submitted work. All other authors declare that they have no competing interests.